L-type Channels Research Articles

L-type calcium ion channel-mediated activation of autophagy in vascular smooth muscle cells via thonningianin A (TA) alleviates vascular calcification in type 2 diabetes mellitus

Vascular calcification (VC) is associated with increased morbidity and mortality, especially among people with type 2 diabetes mellitus (T2DM). The pathogenesis of vascular calcification is incompletely understood, and until now, there have been no effective therapeutics for vascular calcification. The L-type calcium ion channel in the cell membrane is vital for Ca2+ influx. The effect of L-type calcium ion channels on autophagy remains to be elucidated. Here, the natural compound thonningianin A (TA) was found to ameliorate vascular calcification in T2DM via the activation of L-type calcium ion channels. The results showed that TA had a concentration-dependent ability to decrease the transcriptional and translational expression of the calcification-related proteins runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteopontin (OPN) (P < 0.01) via ATG7-dependent autophagy in β-glycerophosphate (β-GP)- and high glucose (HG)-stimulated primary mouse aortic smooth muscle cells (MASMCs) and alleviate aortic vascular calcification in VitD3-stimulated T2DM mice. However, nifedipine, an inhibitor of L-type calcium ion channels, reversed TA-induced autophagy and Ca2+ influx in MASMCs. Molecular docking analysis revealed that TA was located in the hydrophobic pocket of Cav1.2 α1C and was mainly composed of the residues Ile, Phe, Ala and Met, which confirmed the efficacy of TA in targeting the L-type calcium channel of Cav1.2 on the cell membrane. Moreover, in an in vivo model of vascular calcification in T2DM mice, nifedipine reversed the protective effects of TA on aortic calcification and the expression of the calcification-related proteins RUNX2, BMP2 and OPN (P < 0.01). Collectively, the present results reveal that the activation of cell membrane L-type calcium ion channels can induce autophagy and ameliorate vascular calcification in T2DM. Thonningianin A (TA) can target and act as a potent activator of L-type calcium ion channels. Thus, this research revealed a novel mechanism for autophagy induction via L-type calcium ion channels and provided a potential therapeutic for vascular calcification in T2DM.

Inhibitory effect and underlying mechanism of essential oil of Prangos ferulacea Lindl (L.) on spontaneous and induced uterine contractions in non-pregnant rats

Evidence suggests the use of natural compounds as support in the management of uterine contractility disorders. We recently demonstrated that the essential oil of Apiacea Prangos ferulacea (L.) (Prangoil) modulates intestinal smooth muscle contractility. Thus, we aimed to evaluate if Prangoil could also affect the contractility of uterine muscle in non-pregnant rat and to investigate the related action mechanism/s. The effects of the aromatic monoterpenes, β-ocimene and carvacrol, constituents of Prangoil, were also evaluated. Spontaneous contractions and contraction-induced by K+-depolarization and oxytocin in rat uterus were recorded in vitro, using organ bath technique. Prangoil reduced the amplitude of spontaneous contractions as well as responses to KCl and oxytocin. β-ocimene and carvacrol matched oil inhibitory effects. Prangoil effects were not affected by nitrergic and adenylyl cyclase inhibitors or non-specific potassium channel blocker, but they were reduced by nifedipine, L-type calcium channel inhibitor, or 2-aminoethoxydiphenylborate (2-APB), membrane-permeant inositol 1,4,5-triphosphate receptor inhibitor. The response to β-ocimene was reduced by nifedipine and by 2-APB (20 μM), whilst carvacrol inhibitory effect was attenuated only by nifedipine. In conclusion, Prangoil, and its components, β-ocimene and carvacrol, reduced spontaneous and KCl or oxytocin-induced contractions of rat myometrium, mainly modulating extracellular Ca2+ influx through L-Type channels and Ca2+ release from the intracellular store. Further studies could contribute to evaluate the potential use of Prangoil against disorders characterized by abnormal uterine contractions.

Quinpirole inhibits levodopa-induced dyskinesias at structural and behavioral levels: Efficacy negated by co-administration of isradipine

Dopamine depletion associated with parkinsonism induces plastic changes in striatal medium spiny neurons (MSN) that are maladaptive and associated with the emergence of the negative side-effect of standard treatment: the abnormal involuntary movements termed levodopa-induced dyskinesia (LID). Prevention of MSN dendritic spine loss is hypothesized to diminish liability for LID in Parkinson's disease. Blockade of striatal CaV1.3 calcium channels can prevent spine loss and significantly diminish LID in parkinsonian rats. While pharmacological antagonism with FDA approved CaV1 L-type channel antagonist dihydropyridine (DHP) drugs (e.g, isradipine) are potentially antidyskinetic, pharmacologic limitations of current drugs may result in suboptimal efficacy. To provide optimal CaV1.3 antagonism, we investigated the ability of a dual pharmacological approach to more potently antagonize these channels. Specifically, quinpirole, a D2/D3-type dopamine receptor (D2/3R) agonist, has been demonstrated to significantly reduce calcium current activity at CaV1.3 channels in MSNs of rats by a mechanism distinct from DHPs. We hypothesized that dual inhibition of striatal CaV1.3 channels using the DHP drug isradipine combined with the D2/D3 dopamine receptor agonist quinpirole prior to, and in conjunction with, levodopa would be more effective at preventing structural modifications of dendritic spines and providing more stable LID prevention. For these proof-of-principle studies, rats with unilateral nigrostriatal lesions received daily administration of vehicle, isradipine, quinpirole, or isradipine + quinpirole prior to, and concurrent with, levodopa. Development of LID and morphological analysis of dendritic spines were assessed. Contrary to our hypothesis, quinpirole monotherapy was the most effective at reducing dyskinesia severity and preventing abnormal mushroom spine formation on MSNs, a structural phenomenon previously associated with LID. Notably, the antidyskinetic efficacy of quinpirole monotherapy was lost in the presence of isradipine co-treatment. These findings suggest that D2/D3 dopamine receptor agonists when given in combination with levodopa and initiated in early-stage Parkinson's disease may provide long-term protection against LID. The negative interaction of isradipine with quinpirole suggests a potential cautionary note for co-administration of these drugs in a clinical setting.

Inactivation influences the extent of inhibition of voltage-gated Ca+2 channels by Gem-implications for channelopathies.

Voltage-gated Ca2+ channels (VGCC) directly control muscle contraction and neurotransmitter release, and slower processes such as cell differentiation, migration, and death. They are potently inhibited by RGK GTP-ases (Rem, Rem2, Rad, and Gem/Kir), which decrease Ca2+ channel membrane expression, as well as directly inhibit membrane-resident channels. The mechanisms of membrane-resident channel inhibition are difficult to study because RGK-overexpression causes complete or near complete channel inhibition. Using titrated levels of Gem expression in Xenopus oocytes to inhibit WT P/Q-type calcium channels by ∼50%, we show that inhibition is dependent on channel inactivation. Interestingly, fast-inactivating channels, including Familial Hemiplegic Migraine mutants, are more potently inhibited than WT channels, while slow-inactivating channels, such as those expressed with the Cavβ2a auxiliary subunit, are spared. We found similar results in L-type channels, and, remarkably, Timothy Syndrome mutant channels were insensitive to Gem inhibition. Further results suggest that RGKs slow channel recovery from inactivation and further implicate RGKs as likely modulating factors in channelopathies.

Abstract P3142: Engineering A Genetically Encoded Enhancer Of L-type Ca 2+ Currents

L-type calcium channels (LTCC) play a crucial role as they serve as vital portals for Ca 2+ entry into cardiac myocytes, neurons, and other physiological systems. Altered LTCC function is linked to various pathologies including cardiac arrhythmias and neurodevelopmental disorders. In particular, genetic studies have identified loss-of-function mutations in Ca V 1.2 channels in patients with shortened QT interval and Brugada Syndrome, as well as neuropsychiatric disorders. Given the broad physiological importance of LTCC in various tissues, conventional pharmacological agonists of LTCC may yield off-target effects and toxicity. Therefore, new approaches to upregulate LTCC function in a cell-type dependent manner is sought after. To do so, we here develop a genetically encoded enhancer of calcium channels (GeeCC, “geek”) by leveraging both a cardiac specific L-type channel modulator, leucine rich repeat containing protein 10 (Lrrc10), and selective nanobodies that target the LTCC complex. Specifically, Lrrc10 co-expression markedly upregulates LTCC currents. Furthermore, mutations in Lrrc10 are linked to cardiac diseases, including cardiomyopathies. Through extensive structure-function analysis, we identified a minimal action domain within Lrrc10 that is critical for functional modulation of LTCC. However, as this segment alone has a low affinity for the channel complex, its expression alone yielded minimal changes in Ca 2+ current amplitude. However, fusion of this Lrrc10 domain to a nanobody (nb.F3) that targets the Ca 2+ channel beta subunit resulted in a significant increase in the LTCC current amplitude. In depth analysis revealed exquisite selectivity of GeeCC for Ca V 1.2/Ca V 1.3 channels. In mouse cardiomyocytes, deployment of GeeCC via adenovirus enhances endogenous L-type currents when compared to uninfected controls or cardiomyocytes infected with nanobodies alone. Additionally, in neurons, co-expression of GeeCC enhanced excitation-transcription coupling. In all, we have developed a genetically-encoded approach to selectively enhance L-type currents. This strategy may enable new insights into cardiovascular and neuronal physiology and pathophysiology, and may have potential therapeutic applications.

Resting Membrane Potential Modulates Chemoreceptor Sensitivity in Nematocyst Discharge of the Sea Anemone Exaiptasia diaphena.

AbstractExtracellular calcium has been known to be required for in situ nematocyst discharge for more than 60 years, yet calcium's role in nematocyst discharge is poorly understood. Currently, we know that extracellular calcium plays at least two distinct roles in in situ nematocyst discharge. First, calcium plays a role in the triggering of discharge by physical contact, most likely involving transient receptor potential channels. Second, activated L-type calcium channels desensitize nematocyst discharge predisposed to discharge by stimulated chemoreceptors for N-acetylated sugars, such as N-acetylneuraminic acid (NANA). It is not known whether the stimulated NANA signaling pathway activates L-type channels electrogenically through membrane depolarization or directly by phosphorylation of the channel. We hypothesize that the activated NANA signaling pathway initiates desensitization by depolarizing cell membrane potentials to activate voltage-gated L-type calcium channels. Consistent with our hypothesis, we show that depolarization induced by blocking voltage-gated potassium channels with 4-aminopyridine selectively activates Ca2+ influx into tentacle ectodermal cells via L-type channels and inhibits in situ nematocyst discharge from chemosensitized anemones. Furthermore, preventing membrane depolarization with valinomycin or hyperpolarizing resting membrane potentials with low-potassium seawater suppresses NANA-induced Ca2+ influx, prevents desensitization of in situ nematocyst discharge, and enhances NANA sensitivity. Thus, changing resting membrane potentials modulates NANA sensitivity, and NANA-induced depolarization drives desensitization. We suggest that desensitization of the NANA signaling pathway occurs by a feedback pathway involving calcium channels that are activated by NANA-induced depolarization. Elucidating the desensitization pathway may suggest methods to protect or prevent public health cases of nematocyst stinging.

Decreased nitrosylation of CaMKII causes aging-associated impairments in memory and synaptic plasticity in mice.

CaMKII has molecular memory functions because transient calcium ion stimuli can induce long-lasting increases in its synaptic localization and calcium ion-independent (autonomous) activity, thereby leaving memory traces of calcium ion stimuli beyond their duration. The synaptic effects of two mechanisms that induce CaMKII autonomy are well studied: autophosphorylation at threonine-286 and binding to GluN2B. Here, we examined the neuronal functions of additional autonomy mechanisms: nitrosylation and oxidation of the CaMKII regulatory domain. We generated a knock-in mouse line with mutations that render the CaMKII regulatory domain nitrosylation/oxidation-incompetent, CaMKIIΔSNO, and found that it had deficits in memory and synaptic plasticity that were similar to those in aged wild-type mice. In addition, similar to aged wild-type mice, in which CaMKII was hyponitrosylated, but unlike mice with impairments of other CaMKII autonomy mechanisms, CaMKIIΔSNO mice showed reduced long-term potentiation (LTP) when induced by theta-burst stimulation but not high-frequency stimulation (HFS). As in aged wild-type mice, the HFS-LTP in the young adult CaMKIIΔSNO mice required L-type voltage-gated calcium ion channels. The effects in aged mice were likely caused by the loss of nitrosylation because no decline in CaMKII oxidation was detected. In hippocampal neurons, nitrosylation of CaMKII induced its accumulation at synapses under basal conditions in a manner mediated by GluN2B binding, like after LTP stimuli. However, LTP-induced synaptic CaMKII accumulation did not require nitrosylation. Thus, an aging-associated decrease in CaMKII nitrosylation may cause impairments by chronic synaptic effects, such as the decrease in basal synaptic CaMKII.

Englerin A Inhibits T-Type Voltage-Gated Calcium Channels at Low Micromolar Concentrations.

Englerin A (EA) is a potent agonist of tetrameric transient receptor potential canonical (TRPC) ion channels containing TRPC4 and TRPC5 subunits. TRPC proteins form cation channels that are activated by plasma membrane receptors. They convert extracellular signals such as angiotensin II into cellular responses, whereupon Na+ and Ca2+ influx and depolarization of the plasma membrane occur. Via depolarization, voltage-gated Ca2+ (CaV) channels can be activated, further increasing Ca2+ influx. We investigated the extent to which EA also affects the functions of CaV channels using the high-voltage-activated L-type Ca2+ channel CaV1.2 and the low-voltage-activated T-type Ca2+ channels CaV3.1, CaV3.2, and CaV3.3. After expression of cDNAs in human embryonic kidney (HEK293) cells, EA inhibited currents through all T-type channels at half-maximal inhibitory concentrations (IC50) of 7.5 to 10.3 μM. In zona glomerulosa cells of the adrenal gland, angiotensin II-induced elevation of cytoplasmic Ca2+ concentration leads to aldosterone release. We identified transcripts of low- and high-voltage-activated CaV channels and of TRPC1 and TRPC5 in the human adrenocortical (HAC15) zona glomerulosa cell line. Although no EA-induced TRPC activity was measurable, Ca2+ channel blockers distinguished T- and L-type Ca2+ currents. EA blocked 60% of the CaV current in HAC15 cells and T- and L-type channels analyzed at -30 mV and 10 mV were inhibited with IC50 values of 2.3 and 2.6 μM, respectively. Although the T-type blocker Z944 reduced basal and angiotensin II-induced 24-hour aldosterone release, EA was not effective. In summary, we show here that EA blocks CaV1.2 and T-type CaV channels at low-micromolar concentrations. SIGNIFICANCE STATEMENT: In this study we showed that englerin A (EA), a potent agonist of tetrameric transient receptor potential canonical (TRPC)4- or TRPC5-containing channels and currently under investigation to treat certain types of cancer, also inhibits the L-type voltage-gated Ca2+ (CaV) channel CaV1.2 and the T-type CaV channels CaV3.1, CaV3.2, and CaV3.3 channels at low micromolar concentrations.

#2696 IN SILICO ELECTROPHYSIOLOGICAL STUDY REVEALS TAMSULOSIN MEDIATE URETER SMOOTH MUSCLE CONTRACTION BY INCREASING POTASSIUM CURRENT

Abstract Background and Aims Tamsulosin is a common α1-AR antagonist that is prescribed to patients with acute kidney stone episodes [Scotland et al., 2021]. According to accumulated experimental data, abnormal peristaltic contraction of the ureter smooth muscle (USM) causes acute kidney stone episodes [Lang et al., 2006]. Intracellular electrical activities like membrane depolarization and action potentials play important roles in modulating the USM contraction by releasing intracellular calcium from the sarcoplasmic reticulum. Therefore, an electrophysiological study will be helpful to assess the USM cell's electrical activities and in diagnosing abnormal USM contraction. Transient potassium channels (KA) are prevalent in different systems and hold immense importance for maintaining/performing selective electrophysiological functions. The present study investigates the altered electrophysiological characteristics of the USM cell because of the induction of Tamsulosin doses. Method The single isolated USM cell comprises several voltage-gated ion channels, such as two voltage-gated calcium (T-type, and L-type) channels, one voltage-gated fast potassium (KA) channel, one calcium-dependent large conductance potassium channel, and an HCN channel. The individual ion channel currents and the membrane potential are recorded by utilizing both the voltage clamp and current clamp protocols. A Tamsulosin drug (0.5 mg) model for the KA channel is introduced by multiplying the maximal conductance of the KA channel with a scaling factor between 0 and 1 to mimic the drug concentration. Statistical analysis protocols are evaluated for analytical purposes. Results In this in silico electrophysiological model, the resting membrane potential is maintained at −75 mV, as this value is cited by various experimental studies. The ionic concentrations and biophysical parameters for all ion channels are varied in the physiological ranges. Under the voltage clamp protocol, the voltage steps are increased from a holding potential of −80 mV to −10 mV with a 10 mV of step voltage. In Figure 1, the red and blue solid lines represent recorded outward KA current from the isolated USM cell under both control and applied Tamsulosin dose conditions. Figure 1 reveals that the KA current is elevated after the application of the Tamsulosin drug regarding all step voltages. As a result, the total outward current is elevated and the USM action potential duration is shortened. It is also observed that the steady-state activation curve of the KA channel is shifted to more negative after applying Tamsulosin. The USM cell is electrically less excitable with the Tamsulosin. Conclusion This first in-silico study reveals the KA ionic current, which plays a major role in coordinating ureteral contractions, is altered due to Tamsulosin. It implies that the pharmacological maneuver of Tamsulosin is substantially beneficial to treat dysfunctional peristalsis by reducing the symptoms and installation complexities from the ureteral stents procedure.

Fluoride induces neutrophil extracellular traps and aggravates brain inflammation by disrupting neutrophil calcium homeostasis and causing ferroptosis

Endemic fluorosis (EF) has been listed as one of the serious public health problems in many countries. Long-term exposure to high fluoride can lead to severe neuropathological damage to the brain. Although long-term research has revealed the mechanism of some brain inflammation caused by excessive fluoride, the role of intercellular interactions, especially immune cells, in brain damage is still unclear. Fluoride can induce ferroptosis and inflammation in the brain in our study. A co-culture system of neutrophil extranets and primary neuronal cells showed that fluoride can aggravate neuronal cell inflammation by causing neutrophil extranets (NETs).In terms of the mechanism of action, we found that fluoride leads to the opening of calcium ion channels by causing neutrophil calcium imbalance, which in turn leads to the opening of L-type calcium ion channels (LTCC). Extracellular free iron enters the cell from the open LTCC, leading to neutrophil ferroptosis, which releases NETs. Blocking LTCC (nifedipine) rescued neutrophil ferroptosis and reduced the generation of NETs. Inhibition of ferroptosis (Fer-1) did not block cellular calcium imbalance. In summary, our study explores the role of NETs in fluoride-induced brain inflammation and suggests that blocking calcium channels may be one of the possibilities to rescue fluoride-induced ferroptosis.

Synthesis of 6-alkoxy and 6-hydroxy-alkyl amine derivatives of braylin as vasorelaxing agents

Braylin (10b) is a 8,8-dimethyl chromenocoumarin present in the plants of the family Rutaceae and Meliaceae and possesses vasorelaxing and anti-inflammatory activities. In this study, six 6-alkoxy (10b, 15–19), and twelve 6-hydroxy-alkyl amine (20a-20l) derivatives of braylin (11 and 12) were synthesized to delineate its structural requirement for vasorelaxing activity. The synthesized compounds were evaluated for vasorelaxation response in preconstricted intact rat Main Mesenteric Artery (MMA). The compounds showed l-type VDCC channel blockade depended and endothelium-independent vasorelaxation within the range of Emax < 50.00–96.70 % at 30 µM. Amongst all, 6-alkoxy derivatives were more active than 6-hydroxy-alkyl amine derivatives. The structural refinements about braylin showed that deletion of its methoxy group or homologation beyond ethoxy group presented deleterious effect on vasorelaxation response of braylin. Interestingly, substituting the ethoxy group in 10b presented the best activity and selectivity towards l-type VDCC channel blockade, a specific target cardiovascular function.

Simulations of active zone structure and function at mammalian NMJs predict that loss of calcium channels alone is not sufficient to replicate LEMS effects.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune-mediated neuromuscular disease thought to be caused by autoantibodies against P/Q-type voltage-gated calcium channels (VGCCs), which attack and reduce the number of VGCCs within transmitter release sites (active zones; AZs) at the neuromuscular junction (NMJ), resulting in neuromuscular weakness. However, patients with LEMS also have antibodies to other neuronal proteins, and about 15% of patients with LEMS are seronegative for antibodies against VGCCs. We hypothesized that a reduction in the number of P/Q-type VGCCs alone is not sufficient to explain LEMS effects on transmitter release. Here, we used a computational model to study a variety of LEMS-mediated effects on AZ organization and transmitter release constrained by electron microscopic, pharmacological, immunohistochemical, voltage imaging, and electrophysiological observations. We show that models of healthy AZs can be modified to predict the transmitter release and short-term facilitation characteristics of LEMS and that in addition to a decrease in the number of AZ VGCCs, disruption in the organization of AZ proteins, a reduction in AZ number, a reduction in the amount of synaptotagmin, and the compensatory expression of L-type channels outside the remaining AZs are important contributors to LEMS-mediated effects on transmitter release. Furthermore, our models predict that antibody-mediated removal of synaptotagmin in combination with disruption in AZ organization alone could mimic LEMS effects without the removal of VGCCs (a seronegative model). Overall, our results suggest that LEMS pathophysiology may be caused by a collection of pathological alterations to AZs at the NMJ, rather than by a simple loss of VGCCs.NEW & NOTEWORTHY We used a computational model of the active zone (AZ) in the mammalian neuromuscular junction to investigate Lambert-Eaton myasthenic syndrome (LEMS) pathophysiology. This model suggests that disruptions in presynaptic active zone organization and protein content (particularly synaptotagmin), beyond the simple removal of presynaptic calcium channels, play an important role in LEMS pathophysiology.

Nanomaterial's interfacial stimulation of vascular endothelial cells and divergent guidances for nanomedicine treating vasculature-associated diseases

Blood vasculature plays an essential role in transporting nanomaterials in vivo. However, the interaction between vascular endothelial cells (VECs) and nanomaterials lacked deep understanding, impeding the development of safe and efficient nanomedicine. Here, we discover that VECs can be significantly stimulated by two-dimensional PEGylated lamellar materials (PLMs) without being internalized. Additional experiments and computational simulations suggest that the unique VEC stimulation can be ascribed to the interaction between the PLMs and the membrane lipids, which induces ion-transport-related conformational change of L-type calcium ion channel Cav1.3. Additionally, the in vivo studies demonstrate that PLMs-stimulated VECs have divergent impacts on two types of vasculature-associated diseases: exacerbating the development of atherosclerosis while magnifying the accumulation of nanomedicine within tumors. Thus, the current study not only advances our understanding of how specific proteins on VEC membranes could be stimulated by PLMs but also guides the rational use of promising PLMs in nanomedicine.

Non-prophylactic resveratrol-mediated protection of neurite integrity under chronic hypoxia is associated with reduction of Cav1.2 channel expression and calcium overloading.

Cellular hypoxia is a major cause of oxidative stress, culminating in neuronal damage in neurodegenerative diseases. Numerous ex vivo studies have implicated that hypoxia episodes leading to disruption of Ca2+ homeostasis and redox status contribute to the progression of various neuropathologies and cell death. Isolation and maintenance of primary cell culture being cost-intensive, the details of the time course relationship between Ca2+ overload, L-type Ca2+ channel function, and neurite retraction under chronic and long-term hypoxia remain undefined. In order to explore the effect of oxidative stress and Ca2+ overload on neurite length, first, we developed a 5-day-long neurite outgrowth model using N2a cell line. Second, we propose a chronic hypoxia model to investigate the modulation of the L-type Ca2+ channel (Cav1.2) and oxidative resistance gene (OXR1) expression level during the process of neurite retraction and neuronal damage over 32h. Thirdly, we developed a framework for quantitative analysis of cytosolic Ca2+, superoxide formation, neurite length, and constriction formation in individual cells using live imaging that provides an understanding of molecular targets. Our findings suggest that an increase in cytosolic Ca2+ is a feature of an early phase of hypoxic stress. Further, we demonstrate that augmentation in the L-type channel leads to amplification in Ca2+ overload, ROS accumulation, and a reduction in neurite length during the late phase of hypoxic stress. Next, we demonstrated that non-prophylactic treatment of resveratrol leads to the reduction of calcium overloading under chronic hypoxia via lowering of L-type channel expression. Finally, we demonstrate that resveratrol-mediated reduction of Cav1.2 channel and STAT3 expression are associated with retention of neurite integrity. The proposed in vitro model assumes significance in the context of drug designing and testing that demands monitoring of neurite length and constriction formations by imaging before animal testing.

Aluminum alters excitability by inhibiting calcium, sodium, and potassium currents in bovine chromaffin cells.

Aluminum (Al3+ ) has long been related to neurotoxicity and neurological diseases. This study aims to describe the specific actions of this metal on cellular excitability and neurotransmitter release in primary culture of bovine chromaffin cells. Using voltage-clamp and current-clamp recordings with the whole-cell configuration of the patch clamp technique, online measurement of catecholamine release, and measurements of [Ca2+ ]c with Fluo-4-AM, we have observed that Al3+ reduced intracellular calcium concentrations around 25% and decreased catecholamine secretion in a dose-dependent manner, with an IC50 of 89.1μM. Al3+ blocked calcium currents in a time- and concentration-dependent manner with an IC50 of 560 μM. This blockade was irreversible since it did not recover after washout. Moreover, Al3+ produced a bigger blockade on N-, P-, and Q-type calcium channels subtypes (69.5%) than on L-type channels subtypes (50.5%). Sodium currents were also inhibited by Al3+ in a time- and concentration-dependent manner, 24.3% blockade at the closest concentration to the IC50 (399 μM). This inhibition was reversible. Voltage-dependent potassium currents were low affected by Al3+ . Nonetheless, calcium/voltage-dependent potassium currents were inhibited in a concentration-dependent manner, with an IC50 of 447 μM. This inhibition was related to the depression of calcium influx through voltage-dependent calcium channels subtypes coupled to BK channels. In summary, the blockade of these ionic conductance altered cellular excitability that reduced the action potentials firing and so, the neurotransmitter release and the synaptic transmission. These findings prove that aluminum has neurotoxic properties because it alters neuronal excitability by inhibiting the sodium currents responsible for the generation and propagation of impulse nerve, the potassium current responsible for the termination of action potentials, and the calcium current responsible for the neurotransmitters release.

The Effects of Mechanical Load on Chondrogenic Responses of Bone Marrow Mesenchymal Stem Cells and Chondrocytes Encapsulated in Chondroitin Sulfate-Based Hydrogel.

Articular cartilage is vulnerable to mechanical overload and has limited ability to restore lesions, which leads to the development of chronic diseases such as osteoarthritis (OA). In this study, the chondrogenic responses of human bone marrow mesenchymal stem cells (BMMSCs) and OA cartilage-derived chondrocytes in 3D chondroitin sulfate-tyramine/gelatin (CS-Tyr)/Gel) hydrogels with or without experimental mechanical load have been investigated. Chondrocytes were smaller in size, had slower proliferation rate and higher level of intracellular calcium (iCa2+) compared to BMMSCs. Under 3D chondrogenic conditions in CS-Tyr/Gel with or without TGF-β3, chondrocytes more intensively secreted cartilage oligomeric matrix protein (COMP) and expressed collagen type II (COL2A1) and aggrecan (ACAN) genes but were more susceptible to mechanical load compared to BMMSCs. ICa2+ was more stably controlled in CS-Tyr/Gel/BMMSCs than in CS-Tyr/Gel/chondrocytes ones, through the expression of L-type channel subunit CaV1.2 (CACNA1C) and Serca2 pump (ATP2A2) genes, and their balance was kept more stable. Due to the lower susceptibility to mechanical load, BMMSCs in CS-Tyr/Gel hydrogel may have an advantage over chondrocytes in application for cartilage regeneration purposes. The mechanical overload related cartilage damage in vivo and the vague regenerative processes of OA chondrocytes might be associated to the inefficient control of iCa2+ regulating channels.

Phosphodiesterase 1 inhibition underlies long QT phenotype caused by the human N98S-calmodulin mutant.

We previously demonstrated that mice heterozygous for the p.N98S mutation in the calmodulin (CaM)-encoding Calm1 gene (Calm1N98S/+) exhibit β-adrenergic hyperresponsiveness of cardiac L-type current (ICa.L). We hypothesized that N98S-CaM-mediated inhibition of Ca2+/CaM-dependent phosphodiesterase (PDE1) increases cAMP-signaling at the L-type channel, amplifying the effect of β-adrenergic stimulation on ICa.L. Peak ICa.L densities were significantly larger (−13.9±0.6 pA/pF vs. −10.3±0.3 pA/pF;mean±SEM at 0 mV) and ICa.L activated at more negative potentials (Vhalf:-16.2±0.8 mV vs. -12.9±0.8 mV) in Calm1N98S/+vs. Calm1+/+ ventricular myocytes (VMs) treated with the β-adrenergic agonist isoproterenol (ISO; 5 nM;P = 0.005; 21 cells/genotype). Substitution of external Ca2+ with Ba2+ plus dialysis of VMs with 5 μM ryanodine, 1 μM thapsigargin and 10 mM BAPTA abrogated ICa.L potentiation, indicative of its Ca2+-dependence. PDE1-mediated, i.e., Ca2+-sensitive, cAMP hydrolysis was absent in Calm1N98S/+ VM lysates, whereas it contributed 31±3% to the total PDE catalytic activity in Calm1+/+ lysates (n = 6/genotype). The PDE1 inhibitor (PDE1i) Lu-AF50827 (200 nM) eliminated PDE1 activity from and replicated ISO-induced ICa.L potentiation in Calm1+/+ myocytes but had no additional effect on peak ICa.L of ISO-stimulated Calm1N98S/+ myocytes. The protein kinase A (PKA) inhibitor Rp-cAMPS abrogated ISO-evoked ICa.L potentiation of Calm1N98S/+ VMs, indicating that PKA overactivity is both necessary and sufficient for ICa.L potentiation. ISO did not prolong action potential duration at 90% repolarization (APD90) in Calm1+/+ VMs (1 Hz; n = 6), but its combination with PDE1i increased APD90 by 8±2 ms (n = 6; P = 0.005). Despite the larger peak ICa.L density of ISO-stimulated Calm1N98S/+ myocytes, magnitudes of action potential-evoked peak [Ca2+]i elevations with ISO were indistinguishable between the two genotypes. We conclude that N98S-CaM-induced PDE1 inhibition amplifies cAMP signaling at the L-type channel, causing PKA-mediated ICa.L potentiation, but does not impact sarcoplasmic reticulum Ca2+ cycling, compromising feedback inhibition of L-type channels and prolonging APD.

Disease-associated calmodulin variants impair regulation of neuronal voltage-gated calcium channels.

Calmodulin (CaM) is a regulatory protein that associates with numerous binding partners, and it participates in key mechanisms by which multiple types of excitable cells sense and respond to changing levels of calcium. In neurons and cardiomyocytes, calcium ions flux into the cytosol through voltage-gated calcium channels (VGCCs) and bind to CaM. These binding events trigger an array of important feedback processes, including calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF) of multiple VGCC subtypes. In this way, CaM modulates VGCC activity and participates in calcium signaling cascades. Calmodulinopathies are a group of related disorders that arise from pathogenic mutations in CaM. These mutations alter cardiac calcium dynamics in a way that leads to debilitating disorders, including long-QT syndrome (LQTS). LQTS in calmodulinopathy patients has been shown to arise from the mutant forms of CaM preventing the protein from mediating proper CDI of cardiac CaV1.2 L-type channels. However, CaM regulation of VGCCs is not exclusive to the heart, and disruption of these processes in neuronal VGCCs may contribute to the comorbid neurological symptoms frequently observed in calmodulinopathy patients, including autism spectrum disorder, developmental delay, and seizures. We investigated the impacts of calmodulinopathic mutations on VGCCs known to be important for neuronal function. We found that CaM mutants caused diminished CDI in CaV1.3 channels similar to the deficit of CDI seen in CaV1.2 channels, as well as disruption of CDF in CaV2.1 channels. Interestingly, the pattern of CaV2.1 CDF disruptions by certain mutations does not entirely match the pattern seen in CaV1.2 and CaV1.3, indicating a differential effect of select mutations in this neuronal channel. Overall, our results suggest that calmodulinopathic mutations may have consequences to calcium dynamics that extend into neural tissues, in addition to their well-described impacts on cardiac health.

The coupling between calcium channel activity/calcium oscillation and neuritogenesis in cortical neurons.

L-type CaV1 channels transduce membrane excitability into dynamic Ca2+signals, such as autonomous Ca2+ oscillations, which regulate gene transcription in the nucleus critical to neurite outgrowth. Such signaling axis is defined as the coupling between CaV1 activity or Ca2+ oscillation and neurite outgrowth (i.e., CaV1-neuritogenesis coupling). In cortical neurons, stand-alone CaV1-encoded DCT (distal C-terminus) peptides are unveiled to inhibit the CaV1-neuritogenesis coupling, in contrast to the documented role of DCT as nuclear transcription factors to promote neurite outgrowth. When present in the cytosol, DCT peptides downregulate CaV1 activity, Ca2+ influx, pCREB, c-Fos, etc. Importantly, the potency of inhibition is tightly correlated with the affinity of DCT binding CaV1, quantitatively demonstrated by a series of DCT peptides of native origins (Yang 2022 Communications Biology). Autonomous Ca2+ oscillations play key roles in neural development. Specific CaV1 antagonists including DCT peptides are able to abolish oscillations, leading to their effects on neuritogenesis. Aiming at the Ca2+ oscillation-neuritogenesis coupling, with neuron-compatible GCaMP-X that our lab recently developed, we have achieved chronic GCaMP-X imaging which overcomes the toxicity of long/high expression known to conventional GCaMP. Chronic GCaMP-X imaging (∼1 month) of virus-infected cortical neurons recapitulated the tight correlation between spontaneous Ca2+ activity and neuronal development/health with quantitative insights. In support, in vivo imaging from whisker responses and spontaneous Ca2+ slow waves in live mice also demonstrated that GCaMP-X causing less toxicity outperformed GCaMP (Geng 2022 eLife).In summary, the CaV1 activity/Ca2+ oscillation-neuritogenesis coupling and its regulations/perturbations have been examined in vitro and in vivo by DCT peptides and GCaMP-X probes (versus GCaMP), both of which are expected to promote methodology development as well as mechanistic understanding.

Towards next-generation optogenetic actuators of voltage-gated Ca2+ channels.

CaV1 channels (CaV1.2/1.3) are fundamentally involved in the normal function and pathophysiology of the heart and the brain. These channels convey Ca2+ influx that sculpts action potentials, triggers muscle contraction, and mediates excitation-transcription coupling. To orchestrate these diverse functions, CaV1 activity is fine-tuned by multiple regulatory processes, including Ca2+-dependent inactivation (CDI), which is mediated by the Ca2+-binding protein, calmodulin (CaM). Disruption of CaM regulation is linked to cardiac arrhythmias and neurodevelopmental disorders. However, the precise role of this modulation in various physiological settings is not fully understood. In part, this gap in understanding stems from a lack of approaches to dynamically alter CDI. Here, we engineer a novel optogenetic tool to acutely prevent CDI. To do so, we undertook a biomimetic approach by leveraging naturally occurring CaV1 regulatory mechanisms that modify CDI. Specifically, SH3 and cysteine-rich-domain (stac) protein was recently found to selectively prevent CaV1 CDI. Our previous work identified a short amino acid segment of stac called the U-domain that strongly inhibits CDI. Here, we attached the stac3 U-domain to a light-sensitive Lov2 domain to optically switch its accessibility for modulating CaV1 channels, yielding an actuator known as LovU-CaV1. Whole-cell electrophysiological recordings showed that co-expression of LovU-CaV1 resulted in low basal inhibition of CDI of CaV1.2. However, upon light activation of LovU-CaV1, we observed a rapid reduction in CDI that was then reversible. LovU-CaV1 provides a new avenue to optically manipulate endogenous Ca channels in both the heart and the brain. Future studies will employ this tool to dissect the importance of L-type channel inactivation in both cardiomyocytes and in neurons.